The aim of this study was to evaluate the effects of Ascorbic Acid (Vit C) on mean arterial blood pressure (MAP), heart rate (HR) and renal sympathetic nerve activity (RSNA) in renovascular hypertensive rats. Moreover, we compared alterations in the mRNA expression of Neuronal Nitric Oxide Sintase (nNOS), Angiotensin receptor (AT-1) and interleukin 1â (IL-1â) in the brainstem. Experiments were performed in male Wistar rats (6 weeks after renal surgery - Goldblatt hypertension model - 2K-1C). Animals were divided in control (C, n= 6) and hypertensive group (GII, n= 6). Vit C was infused (250 mg/kg, IV) over a 6 minutes infusion period and MAP, HR and RSNA were monitored along 60 minutes. The mRNA expression of nNOS, AT-1 and IL-1â were analyzed in the brainstem tissue by RT-PCR technique. Acute infusion of Vit C in 2K-1C produced a significant decrease in MAP from 157 ± 7 to 120 ± 19 mmHg in the first minute after infusion. No changes in HR were observed. The reduction in MAP was followed by a significant decrease in RSNA (18 ± 6%). The same Vit C infusion in normotensive rats did not produce any changes in MAP, HR and RSNA. The hypertensive animals presented a significant upregulation in nNOS expression in the brainstem (2.3 AU) compared with Control group (1.3 AU). The AT-1 and IL-1â expression were reduced in hypertensive rats compared with normotensive rats (GII 0.15 vs C 0.81 and GII 0.12 vs C 1.1; p<0.001) respectively. Our results suggest a possible role of oxidative stress in the etiology of renovascular hypertension. Supported by CAPES, CNPq-PRONEX

Osteoporosis is a great medical problem, mainly in post-menopausal women. Nitric Oxide (NO) is involved in atheroprotection and bone metabolism regulation, as well as in the mediation of estrogen effects in females. The aim of this study was to evaluate the role of nitric oxide over the epithelial renal calcium (ECaC) and sodium (ENaC) channels expression in ovariectomized rats. Age matched virgin Wistar rats served as control (C, n=5) and the others rats were divided in ovariectomy (OV, n=5) and ovariectomy + L-NAME (OVN, n=6) groups. The L-NAME (50 mg/Kg/day) was administred by gavage during 14 days, in OVN group, starting on the 30th day after surgery. The mRNA expression levels of ECaC and ENaC were analyzed in the renal tissue by cDNA synthesys and Real Time PCR techniques. The OV group showed an increase in ECaC channel expression when compared with C group (C 0,82±0,02 vs OV 1,1±0,1 arbitrary units, p<0,05) and the OVN group had an overexpression in this channel compared with both other groups (OVN 1,8±0,2 vs OV 1,1±0,1 vs C 0,82±0,02 arbitrary units, p<0,05). The ENaC channel did not show diferences in studied groups. The ovariectomized rats presented an upregulation on ECaC nephron channel showing that the absence of estrogen is followed by an increase in calcium reabsorption. When ovariectomy was combined with L-NAME treatment, there was an overexpression of ECaC channel, suggesting that NO blocking exacerbates the effect of ovariectomy over this nephron transporter. The ENaC expression was not changed by neither ovariectomy nor NO blocking. Financial support: CNPq, FAPESP, CAPES, Pronex.

The aim of this study was to evaluate the role of the rostral ventrolateral medulla (RVLM) in the maintenance of hypertension and renal sympathetic nervous activity (RSNA) in a rat model of chronic renal failure (CRF). We also tested the expression of neuronal isoform of nitric oxide synthase (nNOS) and AT1 mRNA in the brainstem. Experiments were performed in male Wistar rats submitted to CRF by 5/6 nephrectomy. After 4 weeks of CRF induction, animals were anesthetized (urethane 1,4 g/Kg) for blood pressure (MAP), heart rate (HR) and RSNA recordings. Microinjections of Glutamate (10 nmol) into the RVLM increased MAP and RSNA in control rats more than in CRF (C, 46 ± 4 and CRF, 27 ± 4 mmHg; RSNA C 58% ± 6 and CRF 38% ± 4). Kynurenic acid into the RVLM did not change MAP and RSNA in both groups. However, inhibition of RVLM by GABA (0,5 M) decreased MAP in CRF rats more than in control rats (DCRF, 63 ± 8 and DC, 33 ± 3 mmHg). AT1 blockade in the RVLM by Candesartan (0,25; 0,5; 1,0 nmol) caused a pressor response and sympathetic activation in CRF and C. There was a downregulation of AT1 mRNA in CRF (C, 0,66 ± 0,09 and CRF, 0,16 ± 0,03 arbitrary unity-AU) in the brainstem and an increase in the expression of nNOS mRNA (C, 0,7 ± 0,08 and CRF, 1,3 ± 0,06 AU). Taken altogether, the results suggest that the hypertension in CRF rats is dependent of sympathetic nervous system. Apparently, AT1 and glutamatergic receptors in the RVLM are not involved in the sympathetic activation in this model of hypertension. An increase in the oxidative stress in the brainstem is probably involved. Supported by CAPES, CNPq-PRONEX.

Increased intrarenal renin-angiotensin system activity contributes to diabetic nephropathy. ANG II generation in mesangial cells (MC) is increased by high-glucose (HG) exposure. This study assessed the mechanisms involved in the glucose-induced ANG II generation in rat MC. Under basal conditions, MC mainly secreted prorenin. HG decreased prorenin secretion and induced a striking 30-fold increase in intracellular renin activity. After 72 h of HG exposure, only the mRNA levels for angiotensinogen and angiotensin-converting enzyme (ACE) were significantly elevated. However, after shorter periods of 24 h of HG stimulation the mRNA levels of the enzymes prorenin and cathepsin B, besides that for ACE, were significantly increased. The results suggest that the HG-induced increase in ANG II generation in MC results from an increase in intracellular renin activity mediated by at least three factors: a time-dependent stimulation of (pro)renin gene transcription, a reduction in prorenin enzyme secretion, and an increased rate of conversion of prorenin to active renin, probably mediated by cathepsin B. The increase in angiotensinogen mRNA in parallel to increased renin activity indicates that HG also increased the availability of the renin substrate. The consistent upregulation of ACE mRNA suggests that, besides renin, ACE is directly involved in the increased mesangial ANG II generation induced by HG.

Accumulation of HSP70 is related to the cytoprotection. It was evaluated whether hyperosmotic stress induces HSP70 accumulation in LLC-PK1 cells, and protects cells against toxicity provoked by cisplatin (Cis) and cyclosporine A (CyA). Cells were maintained in isosmotic (Iso) or hyperosmotic (H) culture medium for 24 h and then exposed to Cis or CyA for an additional period of 12 or 24 h (groups H+Cis and H+CyA). The H medium did not induce cell death and increased both HSP70 mRNA and protein levels, suggesting a role in cell adaptation to H condition. H medium produced partial cytoprotection against Cis and CyA compared with control cells. Despite the cytoprotection, there was a reduction in HSP70 mRNA and protein levels in H+Cis group. In contrast, the H+CyA group presented high levels of HSP70 mRNA and protein. The induction of HSP70 by H medium was associated with tolerance of LLC-PK1 cells against Cis and CyA cytotoxicity but this protection was induced by different mechanisms and depended on the characteristics of the drug used.

Objective To evaluate the effect of an aqueous extract of Phyllanthus niruri (Pn), a plant used in folk medicine to treat lithiasis, on the urinary excretion of endogenous inhibitors of lithogenesis, citrate, magnesium and glycosaminoglycans (GAGs). Materials and methods The effect of chronic (42 days) administration of Pn (1.25 mg/mL/day, orally) was evaluated in a rat model of urolithiasis induced by the introduction of a calcium oxalate (CaOx) seed into the bladder of adult male Wistar rats. The animals were divided into four groups: a sham control (16 rats); a control+Pn (six); CaOx+water instead of Pn (14); and CaOx+Pn (22). Plasma and urine were collected after 42 days of treatment for biochemical analysis and the determination of urinary excretion of citrate, magnesium and GAGs. The animals were then killed and the calculi analysed. Results The creatinine clearance or urinary and plasma concentrations of Na+, K+, Ca2+, oxalate, phosphate and uric acid were unaffected by Pn or the induction of lithiasis. Treatment with Pn strongly inhibited the growth of the matrix calculus and reduced the number of stone satellites compared with the group receiving water. The calculi were eliminated or dissolved in some treated animals (three of 22). The urinary excretion of citrate and magnesium was unaffected by Pn treatment. However, the mean (sd) urinary concentration of GAGs was significantly lower in rats treated with CaOx+Pn, at 5.64 (0.86) mg/g creatinine, than when treated with CaOx + water, at 11.78 (2.21) mg/g creatinine. In contrast, the content of GAGs in the calculi was higher in the CaOx + Pn rats, at 48.0 (10.4) g/g calculus, than in the CaOx + water group, at 16.6 (9.6) g/g calculus. Conclusion These results show that Pn has an inhibitory effect on crystal growth, which is independent of changes in the urinary excretion of citrate and Mg, but might be related to the higher incorporation of GAGs into the calculi.

Reduced bone mineral density (BMD) and an increased risk of vertebral fracture have been reported in calcium-stone-forming (CSF) patients presenting with idiopathic hypercalciuria. We investigated the association between BsmI vitamin D receptor (VDR) polymorphism and BMD in 68 hypercalciuric CSF patients (35 males and 33 premenopausal females, mean age ± SD = 39 ± 10 years). BMD was measured at lumbar spine (L2–L4) and femur neck sites using dual energy X-ray absorptiometry. A 72-hour dietary record and a 24-hour urine sample were obtained from each patient to determine calcium intake and excretion. The allelic frequency found for the sample as a whole was 16% BB, 44% Bb and 40% bb. Mean BMD values did not significantly differ among BB, Bb and bb patients at L2–L4 (1.162 ± 0.10, 1.133 ± 0.11 and 1.194 ± 0.19 g/cm2, mean ± SD, respectively) or at neck sites (0.920 ± 0.11, 0.931 ± 0.15 and 0.982 ± 0.15 g/cm2, respectively). Calcium intake and excretion were also not significantly different among the three genotypes. Patients were then divided into two groups, normal BMD, T-score ≧–1 (n = 34) and low BMD, T-score <–1 (n = 34), to further evaluate the allele influence on previous bone loss. Despite a trend for a higher mean BMD at spine or neck sites for patients with one or two b alleles when compared to BB patients, the difference did not reach statistical significance. The distribution of BB, Bb and bb genotypes in the low-bone-mass group (15, 47 and 38%, respectively) was similar to that in the normal-bone-mass group (18, 41 and 14%, respectively). These data suggest that BsmI VDR polymorphism does not play an important role in the bone loss seen in hypercalciuric CSF patients.

Renin is an enzyme involved in the stepwise generation of angiotensin II. Juxtaglomerular cells are the main source of plasma renin, but renin activity has been detected in other cell types. In the present study we evaluated the presence of renin mRNA in adult male Wistar rat and mouse (C-57 Black/6) mesangial cells (MC) and their ability to process, store and release both the active and inactive forms of the enzyme. Active renin and total renin content obtained after trypsin treatment were estimated by angiotensinogen consumption analyzed by SDS-PAGE electrophoresis and quantified by angiotensin I generation by HPLC. Renin mRNA, detected by RT-PCR, was present in both rat and mouse MC under basal conditions. Active renin was significantly higher (P<0.05) in the cell lysate (43.5 +/- 5.7 ng h-1 10(6) cells) than in the culture medium (12.5 +/- 2.5 ng h-1 10(6) cells). Inactive prorenin content was similar for the intra- and extracellular compartments (9.7 +/- 3.1 and 3.9 +/- 0.9 ng h-1 10(6) cells). Free active renin was the predominant form found in both cell compartments. These results indicate that MC in culture are able to synthesize and translate renin mRNA probably as inactive prorenin which is mostly processed to active renin inside the cell. MC secrete both forms of the enzyme but at a lower level compared with intracellular content, suggesting that the main role of renin synthesized by MC may be the intracellular generation of angiotensin II.

In order to evaluate the progression of renal disease, Munich-Wistar rats were submitted to 5/6 nephrectomy and given whole-body x- or γ-irradiation with or without remnant kidney protection or were submitted only to remnant kidney irradiation. All groups received a single 6-Gy dose immediately after surgery. Whole-kidney function, glomerular hemodynamics, 24-hour proteinuria and histopathology were assessed 60 days after surgery and irradiation. The irradiated nephrectomized animals presented whole-kidney function parameters comparable to those of normal rats. In addition, they were less hypertensive and had higher hematocrit. They showed glomerular hyperfiltration and hypertension even greater than their respective nephrectomized controls. However, the interrelations among the glomerular filtration determinants were somewhat different in irradiated animals. Their 24-hour proteinuria was significantly lower and the sclerosis index and tubulointerstitial injury score were markedly smaller. Among irradiated animals, the worst sclerosis index was observed in those with a shielded remnant kidney and the best in those without protection of the remnant kidney. This led us to speculate about a possible influence of resident mesangial cells on the early events following renal mass ablation and on the maintenance of subsequent physiopathologic changes. Therefore, radiation undoubtedly provoked a beneficial change in the course of renal disease when the renal mass ablation model was employed. Many factors could have contributed to this favorable feature including lower levels of systemic arterial pressure, less increment in ΔP, diminished proteinuria, and maintenance of tubulointerstitial space integrity. Our data also suggest that development of glomerulosclerosis seems to be determined by events occurring immediately after injury.

The activities of serine endopeptidase, prolyl endopeptidase and neutral endopeptidase were determined in tubular fluid collected from several portions of the rat nephron as well as in urine. The enzyme activities were measured by HPLC using bradykinin (BK) as substrate. Free residual peptides of BK obtained by the action of these enzymes on the locally produced BK were also determined. The endopeptidase activities were found to be present throughout the nephron. Equimolar fragments of BK were detected in the early proximal tubule (Arg 1 -Pro 7 , Phe 8 -Arg 9 , Arg 1 -Gly 4 , Phe 5 -Arg 9 , and BK), late proximal tubule (Arg 1 -Phe 5 , Arg 1 -Pro 7 , Gly 4 -Pro 7 , Gly 4 -Arg 9 , and BK), late distal tubule (Arg 1 -Gly 4 , Phe 5 -Arg 9 , Arg 1 -Phe 5 , Ser 6 -Arg 9 , Gly 4 -Arg 9 , BK, and [des-Arg 9 ]BK) and urine (Phe 8 -Arg 9 , Phe 5 -Arg 9 , Arg 1 -Phe 5 , Ser 6 -Arg 9 , Arg 1 -Pro 7 , Gly 4 -Pro 7 , Gly 4 -Arg 9 , BK, and [des-Arg 9 ]BK). Our data suggest that the endopeptidases and exopeptidases are secreted by the nephron. Early proximal tubules secrete angiotensin converting enzyme and neutral endopeptidase, differing from late distal tubules that produce prolyl endopeptidase, serine endopeptidase, carboxypeptidase, and also neutral endopeptidase. All enzymes detected along the rat nephron were found in the urine. The existence of endopeptidases and carboxypeptidase in the distal nephron may have a potential physiological role in the inactivation of the kinins formed by kallikrein in the kidney and also in the inactivation of additional peptides other than BK.

The effects of recombinant human erythropoietin (rHuEPO)-induced polycythemia on renal function and glomerular hemodynamics were evaluated in Munich-Wistar rats (MW+EPO) before and after infusion of indomethacin; the rHuEPO effects on total renal function were also evaluated in 5/6 nephrectomized (CRF) MW and spontaneously hypertensive rats (MW-CRF+EPO and SHR-CRF+EPO, respectively). In normal MW rats, rHuEPO (300 IU/kg BW, 3 x /week, during 2 weeks) induced elevation in MAP, with maintenance of GFR, paralleled by superficial vasodilatation and elevation in SNGFR, suggesting cortical blood redistribution. These hemodynamic alterations induced by rHuEPO were blunted by indomethacin, suggesting a participation of the vasodilator prostaglandins in the renal compensatory mechanism of polycythemia. Elevation in MAP and reduction in GFR occurred in the MW-CRF+EPO group compared with the group receiving vehicle. In contrast, the SHR-CRF+EPO presented a reduction in MAP and maintenance of GFR, suggesting different rHuEPO effects depending on previous renal function and/or hypertensive state.

Objective Previous analysis of the angiotensin I-converting enzyme (ACE) gene in this laboratory showed that primary mesangial cells in culture are able to express ACE mRNA. Moreover, ACE is produced as an ectoenzyme and as a secreted form of the enzyme, indicating a potential effect of local angiotensin II production on glomerular microcirculation. The aim of this study was to purify and characterize the secreted and intracellular ACE forms from mesangial cells in culture. Methods and results Medium from Wistar rats mesangial cells was collected (third passage), incubated for 20 h with RPMI without fetal bovine serum and concentrated 29 times in an Amicon concentrator. The concentrated medium was submitted to gel filtration on an AcA-34 column and two peaks (ACE1, mol. wt 130 000 and ACE2, 60 000) with ACE on activity Hippuryl-His-Leu and Z-Phe-His-Leu were separated. The mesangial cells were collected and ACE enzyme was extracted using Triton X-114, followed by centrifugation and concentration. The supernatant was submitted to the same chromatography as described above and two peaks with ACE activity (ACEInt1, mol. wt 130 000 and ACEInt2, 68 000) were separated. The purified ACE were inhibited by enalaprilat and captopril, two potent competitive inhibitors of ACE and by EDTA, using Hippuryl-His-Leu as a substrate. The Km values were 2 mM for ACE1 and ACE2 and 3 mM for ACEInt1 and ACEInt2. The enzymes ACE1 and ACE2 presented an optimum pH of 8.0 and ACEInt1 and ACEInt2 an optimum pH of 7.5. Conclusion The activities of full-length wild-type and N-domain ACE were characterized by the ratio of the hydrolysis of Z-Phe-His-Leu/Hippuryl-His-Leu, which was 1 and 4, respectively. The ratios found for ACE1, ACE2, ACEInt1 and ACEInt2 in the present study were similar to those described above, suggesting that mesangial cells, besides showing the presence of intracellular ACE, are able to secret both full-length wild-type ACE and N-domain ACE. Thus, they may potentially have an effect, not only on bradykinin and angiotensin I (ACE wild-type), but also on substance P, luteinizing hormone-releasing hormone and Met-enkephalin to interfere with glomerular haemodynamics and with the renal microcirculation.

The role of catecholamines in the distribution of intrarenal blood flow and in single-nephron glomerular filtration rate (SNGFR) was evaluated in anesthetized Wistar rats by the Hanssen technique. Epinephrine (EPI) and norepinephrine (NOR) were infused to produce elevations of 20-30 mmHg in mean arterial pressure. Superficial and juxtamedullary nephron perfusion and filtration were determined by the presence of Prussian blue dye. In the control group, 100% of the nephrons presented a homogeneous pattern of perfusion and filtration. In contrast, a heterogeneous distribution of the dye was found even in the larger arteries (arciform and radial), indicating variable perfusion and filtration in both superficial and juxtamedullary nephrons. The effects of EPI and NOR were also evaluated in the superficial cortex by the micropuncture technique in two additional groups of Munich-Wistar rats. Mean SNGFR was 27% and 54% lower in the EPI- and NOR-treated groups, respectively. No change in mean intraglomerular hydraulic pressure was observed after EPI or NOR infusion in spite of a highly scattered pattern, indicating an important variability in perfusion along the superficial cortex, and/or different sensitivity of the pre- and post-glomerular arterioles. The present data suggest that EPI and NOR may affect intrarenal hemodynamics by modifying perfusion and filtration in both superficial and juxtamedullary glomeruli and not by shifting blood flow from superficial to juxtamedullary nephrons. The heterogeneous pattern of perfusion was a consequence of differential vasoconstriction along the intrarenal arteries, probably due to different density and/or sensitivity of the adrenergic receptor subtypes present in the intrarenal vascular tree.

Whole-kidney function and glomerular hemodynamics were evaluated after acute (50 mg/kg, iv, in bolus) and short-term chronic (50 mg mg/kg, ip, 5 days) acyclovir (ACV) and short-term chronic ganciclovir (Gan; 30 mg/kg, ip, 5 days) treatment in envolemic Munich-Wistar rats. The evaluation of whole-kidney function of the ACV groups showed a significant reduction in total GFR (0.96 +/- 0.10 to 0.28 +/- 0.02 mL/min in the acute group, P < 0.05, and 1.04 +/- 0.09 to 0.33 +/- 0.04 mL/min in the chronic group, P < 0.05) with a marked increase in total renal vascular resistance (TRVR) (33 +/- 5 to 122 +/- 26 mm Hg.min/mL in the acute group and 28 +/- 3 to 74 +/- 18 mm Hg.min/mL in the chronic group, P < 0.05) and a reduction in RPF (2.29 +/- 0.25 to 0.81 +/- 0.15 mL/min in the acute group and 2.57 +/- 0.36 to 1.30 +/- 0.40 mL/min in the chronic group, P < 0.05). Conversely, urinary flow (V') was unchanged (3.6 +/- 0.4 to 3.6 +/- 0.2 microL/min in the acute group) or elevated (3.7 +/- 0.6 to 6.6 +/- 1.4 microL/min in the chronic group, P < 0.05). The evaluation of glomerular hemodynamics after ACV treatment showed a reduction in single-nephron GFR (SNGFR) (46.4 +/- 5.3 to 26.2 +/- 3.4 nL/min in the acute group and 38.7 +/- 5.7 to 21.1 +/- 5.7 nL/min in the chronic group, P < 0.05), a significant elevation in total arteriolar resistance (RT) (2.90 +/- 0.44 to 4.94 +/- 0.77 x 10(10) dyn.s.cm-5 in the acute group and 3.72 +/- 0.45 to 9.00 +/- 2.40 x 10(10) dyn.s.cm-5 in the chronic group, P < 0.05) and a severe reduction in glomerular plasma flow rate (QA) (152.6 +/- 29.5 to 103.8 +/- 27.8 nL/min in the acute group and 149.1 +/- 29.8 to 68.5 +/- 10.0 nL/min in the chronic group, P < 0.05). However, the glomerular ultrafiltration coefficient, Kf, was changed only in the chronic group (0.1002 +/- 0.0165 to 0.0499 +/- 0.0090 nL/(s.mm Hg), P < 0.05). After Gan treatment, no changes were observed in GFR (1.04 +/- 0.09 to 0.96 +/- 0.08 mL/min, with the maintenance of RPF (2.57 +/- 0.36 to 2.66 +/- 0.34 mL/min) and a nonsignificant reduction in TRVR (28 +/- 3 to 20 +/- 3 mm Hg.min/mL. The short-term Gan treatment also showed a different pattern in glomerular hemodynamics by inducing an elevation in SNGFR (38.7 +/- 5.7 to 50.3 +/- 2.8 nL/min, P < 0.05) with no changes in QA (150 +/- 30 to 135 +/- 22 nL/min) and a mild vasodilation, RT (3.7 +/- 0.5 to 2.7 +/- 0.3 x 10(10) dyn.s.cm-5, P < 0.05) associated with an increment in Kf (0.1002 +/- 0.0165 to 0.2400 +/- 0.0700 nL/(s.mm Hg), P < 0.05). Thus, ACV induced acute renal failure by reducing GFR and SNGFR by an increase in TRVR and RT with a reduction in RPF and QA. Also, after short-term treatment with ACV, a reduction in Kf led to a reduction of SNGFR. On the other hand, Gan treatment did not induce acute renal failure by the adopted techniques.